Bottom Line:
We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females.Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

Objective: To identify the causal gene in a multi-incident U.S. kindred with Parkinson's disease (PD).

Methods: We characterized a family with a classical PD phenotype in which 7 individuals (5 males and 2 females) were affected with a mean age at onset of 46.1 years (range, 29-57 years). We performed whole exome sequencing on 4 affected and 1 unaffected family members. Sanger-sequencing was then used to verify and genotype all candidate variants in the remainder of the pedigree. Cultured cells transfected with wild-type or mutant constructs were used to characterize proteins of interest.

Results: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females. The mutation occurred in a highly conserved amino acid residue and was not observed among 87,725 X chromosomes in the Exome Aggregation Consortium dataset. Sequencing of the RAB39B coding region in 587 familial PD cases yielded two additional mutations (c.428C > G [p.A143G] and c.624_626delGAG [p.R209del]) that were predicted to be deleterious in silico but occurred in families that were not sufficiently informative to assess segregation with disease. Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.

Fig3: Effect of p.G192R on RAB39B expression and localization in SK-N-BE(2)C cells. a Western blot of human neuroblastoma (SK-N-BE(2)C) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no significant difference in protein expression between wild-type and mutant RAB39B (Student's t-test, p > 0.05. b Immunofluorescent microscopy of retinoic acid-differentiated SK-N-BE(2)C cells transfected with wild-type or mutant (p.G192R) GFP-tagged RAB39B. Wild-type protein was seen within the cytoplasm and at the plasma membrane where it co-localized with epidermal growth factor receptor (EGFR; blue arrows). Although mutant RAB39B protein was also apparent in the cytoplasm, it less often co-localized with EGFR (white arrows). The scale bar corresponds to 10 μm. c Immunoblot analysis of fractionated protein extracts performed on SK-N-BE(2)C cells transfected with a vector that was empty (mock), or contained GFP-tagged wild-type or mutant RAB39B. Double asterisks indicate a significant difference (p < 0.01) by Student’s t-test (n = 3 replicates)

Mentions:
We then investigated the effects of RAB39B p.G192R in vitro. In PC12 and SK-N-BE(2)C cells transfected with mutant and wild-type constructs there was no substantial difference in RAB39B protein expression (Figs. 2a and 3a). In NGF-differentiated PC12 cells wild-type RAB39B protein was visualized throughout the cytoplasm of cell bodies and neuritic processes (Fig. 2b), and co-localized with the vesicular marker chromogranin A. However, mutant (p.G192R) RAB39B was largely restricted to cell bodies with negligible amounts of protein evident in neuritic processes. In these experiments there was robust expression of both mutant and wild type RAB39B protein, but cellular phenotype can sometimes differ based on the level of transgene over-expression [10]. Thus we used an alternate vector and method of transfection to over-express RAB39B at lower levels in retinoic acid-differentiated SK-N-BE(2)C cells. Wild type RAB39B protein was frequently visualized within the cytoplasm and at the plasma membrane (co-localized with EGFR; Fig. 3b). However, while mutant RAB39B protein was also abundant in the cytoplasm, it was less frequently observed at the plasma membrane. To quantify these findings we performed immunoblot analysis of fractionated protein extracts from these cells (Fig. 3c). The proportion of membrane-bound to cytosolic RAB39B protein was significantly lower in cells expressing mutant protein than wild type protein (p < 0.01).Fig. 2

Fig3: Effect of p.G192R on RAB39B expression and localization in SK-N-BE(2)C cells. a Western blot of human neuroblastoma (SK-N-BE(2)C) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no significant difference in protein expression between wild-type and mutant RAB39B (Student's t-test, p > 0.05. b Immunofluorescent microscopy of retinoic acid-differentiated SK-N-BE(2)C cells transfected with wild-type or mutant (p.G192R) GFP-tagged RAB39B. Wild-type protein was seen within the cytoplasm and at the plasma membrane where it co-localized with epidermal growth factor receptor (EGFR; blue arrows). Although mutant RAB39B protein was also apparent in the cytoplasm, it less often co-localized with EGFR (white arrows). The scale bar corresponds to 10 μm. c Immunoblot analysis of fractionated protein extracts performed on SK-N-BE(2)C cells transfected with a vector that was empty (mock), or contained GFP-tagged wild-type or mutant RAB39B. Double asterisks indicate a significant difference (p < 0.01) by Student’s t-test (n = 3 replicates)

Mentions:
We then investigated the effects of RAB39B p.G192R in vitro. In PC12 and SK-N-BE(2)C cells transfected with mutant and wild-type constructs there was no substantial difference in RAB39B protein expression (Figs. 2a and 3a). In NGF-differentiated PC12 cells wild-type RAB39B protein was visualized throughout the cytoplasm of cell bodies and neuritic processes (Fig. 2b), and co-localized with the vesicular marker chromogranin A. However, mutant (p.G192R) RAB39B was largely restricted to cell bodies with negligible amounts of protein evident in neuritic processes. In these experiments there was robust expression of both mutant and wild type RAB39B protein, but cellular phenotype can sometimes differ based on the level of transgene over-expression [10]. Thus we used an alternate vector and method of transfection to over-express RAB39B at lower levels in retinoic acid-differentiated SK-N-BE(2)C cells. Wild type RAB39B protein was frequently visualized within the cytoplasm and at the plasma membrane (co-localized with EGFR; Fig. 3b). However, while mutant RAB39B protein was also abundant in the cytoplasm, it was less frequently observed at the plasma membrane. To quantify these findings we performed immunoblot analysis of fractionated protein extracts from these cells (Fig. 3c). The proportion of membrane-bound to cytosolic RAB39B protein was significantly lower in cells expressing mutant protein than wild type protein (p < 0.01).Fig. 2

Bottom Line:
We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females.Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

Objective: To identify the causal gene in a multi-incident U.S. kindred with Parkinson's disease (PD).

Methods: We characterized a family with a classical PD phenotype in which 7 individuals (5 males and 2 females) were affected with a mean age at onset of 46.1 years (range, 29-57 years). We performed whole exome sequencing on 4 affected and 1 unaffected family members. Sanger-sequencing was then used to verify and genotype all candidate variants in the remainder of the pedigree. Cultured cells transfected with wild-type or mutant constructs were used to characterize proteins of interest.

Results: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females. The mutation occurred in a highly conserved amino acid residue and was not observed among 87,725 X chromosomes in the Exome Aggregation Consortium dataset. Sequencing of the RAB39B coding region in 587 familial PD cases yielded two additional mutations (c.428C > G [p.A143G] and c.624_626delGAG [p.R209del]) that were predicted to be deleterious in silico but occurred in families that were not sufficiently informative to assess segregation with disease. Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.